Transport-Mediating Colloidal Pharmaceutical Compounds

ABSTRACT

The invention relates to transport mediator-bonded colloids comprising pharmaceutical substances or fluorescence markers, to a method for the production thereof, and to a pharmaceutical preparation comprising said compounds.

The invention relates to colloids bound to transport mediators that maycomprise medicinal compounds or fluorescent markers, to a process forthe preparation thereof, and to a pharmaceutical formulation containingsuch compounds.

The covalent binding to colloids enables substances to be introduced byphagocytosis into cells of the immune system, which would not be takenup, or if so only in negligible amounts, without such modification. EP 1230 935 A1 describes the chemical binding of medicinally activesubstances to a polysaccharide to form a linker. The uptake ofsubstances by correspondingly specialized cells of thereticulohistiocytic system has been demonstrated for a wide variety ofcolloids and particles. However, the introduction of larger moleculesinto cells of the body that are not specialized in phagocytosis is aproblem. In addition, particles and colloids phagocytosed by macrophagesare very quickly taken up into lysosomes after uptake into the cell,where they are degraded by a variety of lytic enzymes. The enzymaticpotential of lysosomes is high; a wide variety of medicinal compounds isdegraded correspondingly quickly by lysosomal enzymes. Of Chlamydiatrachomatis, it is known that this bacterium is taken up by eukaryoticepithelial cells without being degraded enzymatically in the lysosomes.This uptake can be significantly reduced by the presence of heparins orheparin sulfate. Stephens et al. (Infection and Immunity, March 2000, p.1080-1085, Vol. 68, No. 3) show that this effect is based on blocking ofthe heparin binding domain.

Conversely, the authors show that polystyrene microspheres are taken upinto eukaryotic cells by endocytosis after being coated with heparin.Heparin itself binds to a wide variety of enzymes. Patients having anincreased superoxide dismutase activity in the blood serum often exhibita mutated variant of the enzyme (CHU et al. Arteriosclerosis,Thrombosis, and Vascular Biology. 2006; 26:1985).

The mutated variant (R213G) has a glycine instead of the amino acidarginine on position 213. Therefore, binding of the enzyme to heparin isnot possible. Therefore, the afflicted patients have an increasedactivity for the enzyme, because the enzyme is mainly present in theserum rather than in the cell. For bearers of this genetic defect, thismeans a 2.3 times increased risk of the occurrence of ischemic heartdiseases. EP 083 768 A1 describes direct heparin-protein conjugates inwhich the terminal aldose of heparin is bound to the N-terminal aminogroup of a serpin in order to enhance the effect of serpins on bloodcoagulation and respiratory distress syndromes. After being coupled toheparins, proteins and enzymes are very quickly cleared from the serum.Small proteins of below 70 kDa disappear almost completely alreadyduring the first kidney passage. In addition, a wide variety of proteinsare known to present problems of stability and solubility, which can befavorably influenced by covalent binding to a water-solublepolysaccharide.

Heparin belongs to the group of glucosaminoglycans, which consist oflinear chains of sulfated disaccharide units. Each disaccharide unitconsists of one hexuronic acid each, which is variably composed ofglucuronic or iduronic acid and 2-amino-2-deoxy-D-glucose orN-sulfo-D-glucosamine. Like polysaccharides, heparin andglucosaminoglucans have a free aldehyde group at the terminal end.Heparin occurs intracellularly almost exclusively in mast cells.However, more highly sulfated heparins, or heparin sulfates, are foundalmost everywhere on the cell membranes of higher mammals irrespectiveof the kind of organs.

The anticoagulant effect of heparin is primarily based on its affinityto the serin protease inhibitor antithrombin III. The smallest heparinunit that has such effect on AT III includes 5 saccharide units with a3-O-sulfate group at the glucosamine group. This pentasaccharide canform a heparin/ATIII complex, which inhibits the coagulation factor Xa.Thrombin can also be inhibited by binding to specific heparinstructures, but which are not present on a pentasaccharide, which onlyhas 5 saccharide units. For the inhibition of thrombin, heparincompounds having at least 18 saccharide units are necessary.

The molecular size and degree of sulfatation are of critical importancenot only in the selective effect of heparin on coagulation factors, butalso in the interaction with a wide variety of endogenous cytokines andgrowth factors. The interaction of heparin with the fibroblast growthfactor (FGF) requires a minimum number of 18 glucosaminoglucan unitswith a specific sulfatation of the oxygen atom present at C2. Sulfatedheparins from 8 saccharide units exhibit an inhibitory effect onangiogenesis. For these sulfated octasaccharides, inhibition ofangiogenesis in tumor cells is discussed. At the same time, theseheparins seem to have no effect on blood coagulation. The molecular sizeand chemical properties, such as the number and localization of thesulfate, carboxy and amino groups, have a critical influence on theeffect of heparins. The sulfate groups and, less importantly, thecarboxy groups of the iduronic and glucuronic groups have the effectthat heparin is one of the most strongly electronegatively chargedmolecules in mammals. The molecular size as well as the quantity andposition of the sulfate and carboxy groups produce a specific chargepattern or a specific charge distribution on the heparin molecules. Thespecific charge distribution plays a critical role in the affinity ofthe compounds for procoagulant proteins and proteases, and also for theheparin binding domains of endothelial cells. The transport-mediatingfunction of the heparins, which is displayed on cell membranes, is evenmore strongly dependent on the charge structure of thegluco-saminoglucan. Therefore, the covalent incorporation of heparinsinto water-soluble polysaccharides as transport mediators is essentiallydistinguished from the incorporation of the medicinally activecompounds. If possible, the heparin should be coupled to the colloid insuch a way that a binding site with a defined number of disaccharideunits and charges stereoselectively matches the heparin binding domainof the cells. This means that this segment of the heparin remainschemically unbonded and, in addition, non-sterically hindered by theremaining molecule. As with the specific effect of heparin on ATIII andthrombin, the chain length and number of the sulfated hexuronic acid andaminodeoxyglucose units freely protruding from the polysaccharide are ofgreat importance also in this case. In addition, there are indicationsthat the segments of the heparin molecule relevant to the heparinbinding domains are found rather in the middle of the heparin molecule.The part of the molecule utilized for the described association with thebinding domains is strongly electronegatively charged due to the carboxyand sulfate groups. The blocking or disturbing of thisstereospecifically relevant charge pattern by the covalent binding withthe polysaccharide by means of a linker should be prevented for thesesites if possible.

Being a strictly linear glucosaminoglucan, heparin has functional groupsthat can be utilized for binding to other molecules. In the region ofiduronic and glucuronic acid, carboxy groups are present at C6. Thereare hydroxy groups at C2 and C3 and C1 of the first saccharide unit.Every second saccharide group bears an amino group at the C2 atom. Thisamino group and the carboxy group may be sulfated. Finally, heparinbears an aldehyde group at the terminal end.

It is known that point mutations in some genes coding for proteinsresult in a substitution of diaminomonocarboxylic acids by other aminoacids. In some of these cases (superoxide dismutase), this change of theamino acid sequence is accompanied by a loss of the ability to bind toheparin sulfates, whereby the protein is no longer transported into thecell. These results also demonstrate that the incorporation of heparinsinto macromolecules as transport mediators, i.e., for the purpose ofregulating the passage through cell membranes, is dependent onregioselective conditions on the part of the macromolecule. Here, a lossof amino groups not linked by peptide linkages means loss of the abilityto incorporate the transport mediator heparin. In medical technology, itis frequently tried to prevent the formation of blood clots at implantsby the non-specific covalent bonding to heparin.

For the introduction of medicinally active substances into specificorgans and cell systems of the body, the following conditions must bemet:

-   -   1. The uptake of the bound medicament is also effected in cells        that are not specialized in phagocytosis.    -   2. After the passage through the outer cell membrane, the bound        medicament shall not be taken up in lysosomes, and shall not be        degraded enzymatically.    -   3. The medicament complex, which consists of the medicament        chemically bonded to a transport mediator and/or a colloid,        should be water-soluble and circulate in the blood for a        sufficient period of time.    -   4. The medicament complex should have no influence on blood        clotting.

Surprisingly, it has now been found that bonding of a transport mediatorto a colloid (colloid-active compound) solves the above mentionedproblems and serves, in particular, as a suitable transport system formedicaments and/or fluorescence markers covalently linked thereto. Thisholds, in particular, when the colloid and transport mediator arestereoselectively linked together. In addition, it has been surprisinglyfound that the bonding product can bind to membrane-bound andintracellular binding domains if the stereospecific structures of thetransport mediator/colloid compound remain free for association andbinding to the cellular binding domains.

The present invention relates to a compound of general formula (I)

(T-Z)_(n)—P  (I),

wherein

-   -   T is a transport mediator;    -   P is a colloid-active compound;    -   Z is a first linker by means of which T and P are covalently        linked together; and    -   n is an integer of at least 1;        and wherein the transport mediator T and/or the colloid P bears        m groups -(L-A), wherein    -   A is a medicinally active substance or a fluorescence marker;    -   L is a second linker through which P is covalently linked with        A, or through which T is covalently linked with A; and    -   m is an integer that is 0 or at least 1.

Preferably, the transport mediator T has at least one binding site forassociation to cellular binding domains.

According to the invention, the transport mediators T are distinct fromthe colloids P.

Transport mediators T according to the present invention favor uptakeinto cells.

The transport mediator T is a glycan, more preferably selected from thegroup consisting of sialic acid, polysialic acid, neuraminic acid,N-acetylneuraminic acid, mannose, N-acetylmannose,N-propanolmannosamine, fucose, N-acetylfucose, galactose,N-acetylgalactose, glucose, N-acetylglucose, hexoses, N-acetylhexoses,ceramides, glucose-6-phosphate, mannose-6-phosphate,glucosylphosphatidylinositol, retinic acid, immunoglobulins,monoglycerates, diacylglycerates, sphingomyelin, bisphosphonates,glycoproteins, and glycosaminoglycans.

The glycosaminoglycans or glycosaminoglycan derivatives have proven tobe particularly suitable transport mediators T.

Therefore, in a preferred embodiment, the transport mediator T isselected from the group consisting of heparin and heparin sulfate,especially heparin or heparin sulfate having less than 6 saccharideunits. Heparins having less than 6 saccharide units as transportmediators have the particular advantage that the possibly occurringinduction of autoantibodies can be substantially avoided with theseheparins.

The colloid-active compound P (also simply referred to as “colloid P” inthe following) is preferably selected from the group consisting ofamyloses, amylopectins, acemannans, arabinogalactans, galactomannans,galactoglucomannans, xanthans, carrageenan, starch and modified starch.

The modified starches have proven particularly suitable. Starches can bemodified, for example, by hydroxyalkylation or esterification. Inaddition, the starches may also be aminated, for example, by reductiveamination.

Surprisingly, it has been found that amination of the colloid P by meansof reductive amination can yield aminated colloids, especially modifiedstarches, such as aminated hydroxyethyl starch or aminated carboxymethylstarch, which can be incorporated in the transport mediators with a highstereoselectivity in such a way that the compound obtained is verysimilar to the compounds taken up by body cells from transport mediatorcomplexes.

In a preferred embodiment of the present invention, the linking of thetransport mediator and colloid in effected stereoselectively. Further,it is preferred if the linking of the medicinally active substance orthe fluorescence marker with the colloid and/or the transport mediatoris also effected stereoselectively.

In a preferred embodiment, the colloid P is selected from the groupconsisting of hydroxyalkyl starches, esterified starches, carboxyalkylstarches, hydroxyalkyl carboxyalkyl starch, aminated hydroxyalkylstarch, aminated hydroxyalkyl carboxyalkyl starch and aminatedcarboxyalkyl starch.

Carboxyalkyl starches are preferably selected from carboxymethyl starchand carboxyethyl starch.

Advantageously, other specific units that allow the chemical bonding ofthe medicinally active substance or of the fluorescence marker or of thetransport mediator, for example, biotin, amino acids or units bearingsulfide groups, such as cysteine, can also be incorporated into thecolloids.

More preferably, according to the present invention, colloid P is amodified starch selected from the group consisting of hydroxyethylstarch or aminated hydroxyethyl starch, especially a hydroxyethyl starchthat has been aminated by reductive amination.

The hydroxyalkyl groups in the hydroxyethyl starch (HES) have beenintroduced into the molecule for impeding the enzymatic degradation ofthe starch in the serum and for improving the water solubility. Thedegree of substitution, DS, is defined as the ratio of the total numberof substituted monomer units to the total number of monomer units. Inthe following, a degree of substitution, DS, is stated when substituentsare introduced.

In another embodiment of the present invention, the colloid-activecompound has an average molecular weight of from 20,000 to 800,000daltons, preferably from 25,000 to 500,000 daltons, especially from30,000 to 200,000 daltons.

The degree of substitution, DS, of the modified starches, especiallyhydroxyethyl starch, is preferably from 0.2 to 0.8, especially from 0.3to 0.6.

As medicaments A, all substances may be used that can be incorporated inthe above mentioned colloids and/or transport mediators T through alinker L.

The compounds according to the invention may optionally be linked withmedicinally active compounds or fluorescence markers. Preferably, themedicinally active compound is selected from the group consisting ofantibiotics, chemotherapeutics, cytostatic agents, antigens,oligonucleotides, mediators, false metabolic substrates, analgetics andcytotoxic substances.

The fluorescence markers are preferably selected from the groupconsisting of fluorescein isothiocyanate (FITC), phycoerythrin,rhodamide and 2-amino-pyridine.

In addition to purely medicinally active substances, fluorescencemarkers, for example, fluorescein isothiocyanate, may also betherapeutically employed in connection with the transportmediator/colloid complex. Some tumors are known to expressmembrane-bound binding domains in larger numbers, for example, in orderto gain access to the vascular system (FGF receptors). The marking oftransport mediator/colloid complexes according to the invention specificfor such binding domains with fluorescence markers, such as fluoresceinisothiocyanate (A. N. De Belder, K. Granath: Preparation and Propertiesof fluorescein-labelled dextrans, Carbohydrate Research, 30 (1973)375-378) enables the surgeon to optically identify organ fractionshaving a larger number of cells with such binding domains afterinjection of this compound (near infrared fluorescence imaging, NIRF).

In the compound according to formula (I), (T-Z)_(n)—P, the transportmediator T is covalently linked with the colloid P through a firstlinker group Z. In a preferred embodiment of the present invention, thelinker Z is a functional group selected from carboxylic acid ester,carboxylic acid amides, urethane, ether and amine groups or comprises atleast one such functional group. More preferably, the covalent chemicallinkage of T to P through the linker Z is reversible, i.e., can becleaved again without difficulty, for example, enzymatically.

The second linker L, through which the colloid P is covalently linkedwith the medicinally active substance or fluorescence marker, or throughwhich the transport mediator is covalently linked with the medicinallyactive substance or fluorescence marker, also corresponds to the firstlinker Z in its function and design. For the linker L, it isparticularly advantageous if it can be cleaved off again withoutdifficulty, for example, enzymatically, which causes the medicinallyactive substance and/or the fluorescence marker to be released.

The formation of the linker Z or L can be performed by means of methodsdescribed in the prior art for the formation of carboxylic acid esters,carboxylic acid amides, urethanes, ethers and amines.

In a preferred embodiment, the compound according to the invention isobtainable by a reaction of at least one free

-   -   isocyanate group (—NCO);    -   carboxy group (—COOH);    -   carboxylic acid halide group (—CO-A, with A=Cl, Br or I);    -   alkylenecarboxy group (—(CH₂)_(q)—COOH, with q=1-10);    -   ester group (—COOR with R=organic radical);    -   epoxy group;    -   or nucleophilic leaving group;        of the underlying colloid P with a free    -   hydroxy group (—OH)        of the underlying transport mediator T to form the linker Z,        wherein said colloid P and/or transport mediator T is linked        with m units -(L-A).

In another embodiment of the present invention, the compound accordingto the invention is obtainable by a reaction of at least one free

-   -   hydroxy group (—OH)        of the underlying colloid P with a free    -   isocyanate group (—NCO);    -   carboxy group (—COOH);    -   carboxylic acid halide group (—CO-A, with A=Cl, Br or I);    -   alkylenecarboxy group (—(CH₂)_(q)—COOH, with q=1-10);    -   ester group (—COOR with R=organic radical);    -   epoxy group;    -   or nucleophilic leaving group;        of the underlying transport mediator T to form the linker Z,        wherein said colloid P and/or transport mediator T is linked        with m units -(L-A).

In another embodiment of the present invention, the compound accordingto the invention is obtainable by a reaction of at least one free

-   -   amino group (—NH₂)        of the underlying colloid P with a free    -   isocyanate group (—NCO);    -   carboxy group (—COOH);    -   carboxylic acid halide group (—CO-A, with A=Cl, Br or I);    -   alkylenecarboxy group (—(CH₂)_(q)—COOH, with q=1-10);    -   ester group (—COOR with R=organic radical);    -   epoxy group;    -   or nucleophilic leaving group;        of the underlying transport mediator T to form the linker Z,        wherein said colloid P and/or transport mediator T is linked        with m units -(L-A).

Further, in a preferred embodiment, the compound according to theinvention is obtainable by a reaction of at least one free

-   -   isocyanate group (—NCO);    -   carboxy group (—COOH);    -   carboxylic acid halide group (—CO-A, with A=Cl, Br or I);    -   alkylenecarboxy group (—(CH₂)_(q)—COOH, with q=1-10);    -   ester group (—COOR with R=organic radical);    -   epoxy group;    -   or nucleophilic leaving group;        of the underlying colloid P with a free    -   amino group (—NH₂)        of the underlying transport mediator T to form the linker Z,        wherein said colloid P and/or transport mediator T is linked        with m units -(L-A).

More preferably, the compound according to the invention is obtainableby a reaction of at least one free

-   -   hydroxy group (—OH); or    -   amino group (—NH₂)        of the underlying colloid P with a free    -   isocyanate group (—NCO);    -   carboxy group (—COOH);    -   carboxylic acid halide group (—CO-A, with A=Cl, Br or I);    -   alkylenecarboxy group (—(CH₂)_(q)—COOH, with q=1-10);    -   ester group (—COOR with R=organic radical);    -   epoxy group;    -   or nucleophilic leaving group;        of the underlying transport mediator T to form the linker Z,        wherein said colloid P and/or transport mediator T is linked        with m units -(L-A).

According to the present invention, nucleophilic leaving groups arepreferably selected from the group of halides and tosylates.

Further, the compounds according to the invention can be obtainable bythe reaction of a diamine of general formula II

R¹(—NH₂)₂  (II)

wherein R¹ is selected from

-   -   a single bond;    -   linear or branched, saturated or unsaturated, aliphatic or        alicyclic hydrocarbyl groups with 1 to 22 carbon atoms;    -   aryl, aryl-C₁-C₄-alkyl and aryl-C₂-C₆-alkenyl groups with 5 to        12 carbon atoms in the aryl group, which may optionally be        substituted with C₁-C₆ alkyl and/or C₂-C₆ alkoxy groups; or    -   heteroaryl, heteroaryl-C₁-C₄-alkyl and heteroaryl-C₂-C₆-alkenyl        groups with 3 to 8 carbon atoms in the heteroaryl group and one        or two hetero-atom(s) selected from N, O and S, which may be        substituted with C₁-C₆ alkyl and/or C₂-C₆ alkoxy groups;        with a free functional group of the underlying transport        mediator T and at least one free functional group of the        underlying colloid P, which are independently selected from    -   isocyanate group (—NCO);    -   carboxy group (—COOH);    -   carboxylic acid halide group (—CO-A, with A=Cl, Br or I);    -   alkylenecarboxy group (—(CH₂)_(q)—COOH, with q=1-10);    -   ester group (—COOR with R=organic radical);    -   epoxy group;    -   or nucleophilic leaving group;        to form the linker Z, wherein said colloid P and/or transport        mediator T is linked with m units -(L-A).

Suitable diamines include, for example, 1,2-diaminoethane, 1,2- or1,3-diaminopropane, 1,2-, 1,3- or 1,4-diaminobutane, 1,5-diaminopentane,2,2-dimethyl-1,3-diaminopropane, hexamethylenediamine,1,7-diaminoheptane, 1,8-diaminooctane, trimethyl-1,6-diaminohexane,1,9-diaminononane, 1,10-diaminodecane, 1,12-diaminododecane,1,2-diaminocyclohexane, 1,4-diaminocyclohexane,1,3-cyclohexanebis(methylamine), 1,2-phenylenediamine,1,3-phenylenediamine, 1,4-phenylenediamine, 4,4′-Ethylenedianiline,4,4′-methylenedianiline, 4,4′-diaminostilbene, 4,4′-thiodianiline,4-aminophenyldisulfide, 2,6-diaminopyridine, 2,3-diaminopyridine,3,4-diaminopyridine, 2,4-diaminopyrimidine, 4,5-diaminopyrimidine,4,6-diaminopyrimidine.

In addition, in a further embodiment of the present invention, thecompounds according to the invention can be obtained by a reaction of adial of general formula III

R²(—OH)₂  (III),

wherein R² is selected from

-   -   linear or branched, saturated or unsaturated, aliphatic or        alicyclic hydrocarbyl groups with 2 to 22 carbon atoms;    -   aryl, aryl-C₁-C₄-alkyl and aryl-C₂-C₆-alkenyl groups with 5 to        12 carbon atoms in the aryl group, which may optionally be        substituted with C₁-C₆ alkyl and/or C₂-C₆ alkoxy groups; or    -   heteroaryl, heteroaryl-C₁-C₄-alkyl and heteroaryl-C₂-C₆-alkenyl        groups with 3 to 8 carbon atoms in the heteroaryl group and one        or two hetero-atom(s) selected from N, O and S, which may be        substituted with C₁-C₆ alkyl and/or C₂-C₆ alkoxy groups;        with a free functional group of the underlying transport        mediator T and at least one free functional group of the        underlying colloid P, which are independently selected from    -   isocyanate group (—NCO);    -   carboxy group (—COOH);    -   carboxylic acid halide group (—CO-A, with A=Cl, Br or I);    -   alkylenecarboxy group (—(CH₂)_(q)—COOH, with q=1-10);    -   ester group (—COOR with R=organic radical);    -   epoxy group;    -   or nucleophilic leaving group;        to form the linker Z, wherein said colloid P and/or transport        mediator T is linked with m units -(L-A).

Suitable diols include, for example, ethylene glycol, propylene glycol,butylene glycol, and neopentylglycol,pentanediol-1,5,3-methylpentanediol-1,5, bisphenol A, 1,2- or1,4-cyclohexanediol, caprolactonediol (reaction product of caprolactoneand ethylene glycol), hydroxyalkylated bisphenols, trimethylolpropane,trimethylolethane, pentaerythritol, hexanediol-1,6, heptanediol-1,7,octanediol-1,8, butanediol-1,4,2-methyloctanediol-1,8, nonanediol-1,9,decanediol-1,10, cyclohexanedimethylol, di-, tri- and tetraethyleneglycol, di-, tri- and tetrapropylene glycol, polyethylene andpolypropylene glycols with an average molecular weight of from 150 to15,000.

In another embodiment of the present invention, the compounds accordingto the invention are obtainable by a reaction of a dicarboxylic acid ofgeneral formula IV

R³(—COOH)₂  (IV)

wherein R³ is selected from

-   -   a single bond;    -   linear or branched, saturated or unsaturated, aliphatic or        alicyclic hydrocarbyl groups with 1 to 22 carbon atoms;    -   aryl, aryl-C₁-C₄-alkyl and aryl-C₂-C₆-alkenyl groups with 5 to        12 carbon atoms in the aryl group, which may optionally be        substituted with C₁-C₆ alkyl and/or C₂-C₆ alkoxy groups; or    -   heteroaryl, heteroaryl-C₁-C₄-alkyl and heteroaryl-C₂-C₆-alkenyl        groups with 3 to 8 carbon atoms in the heteroaryl group and one        or two hetero-atom(s) selected from N, O and S, which may be        substituted with C₁-C₆ alkyl and/or C₂-C₆ alkoxy groups;        with a free functional group of the underlying transport        mediator T and at least one free functional group of the        underlying colloid P, which are independently selected from    -   amino group (—NH₂); or    -   hydroxy group (—OH)        to form the linker Z, wherein said colloid P and/or transport        mediator T is linked with m units -(L-A),

Suitable dicarboxylic acids include, for example, oxalic acid, malonicacid, succinic acid, glutaric acid, adipic acid, pimelic acid, azelaicacid, sebacic acid, maleic acid, fumaric acid, sorbic acid, phthalicacid, terephthalic acid, isophthalic acid, or agaric acid.

In particular, the compounds according to the invention may also beobtainable by the reaction of a dicarboxylic acid halide of generalformula V

R⁴(—CO-A)₂  (V)

wherein A=Cl, Br or I, and R⁴ is selected from

-   -   a single bond;    -   linear or branched, saturated or unsaturated, aliphatic or        alicyclic hydrocarbyl groups with 1 to 22 carbon atoms;    -   aryl, aryl-C₁-C₄-alkyl and aryl-C₂-C₆-alkenyl groups with 5 to        12 carbon atoms in the aryl group, which may optionally be        substituted with C₁-C₆ alkyl and/or C₂-C₆ alkoxy groups; or    -   heteroaryl, heteroaryl-C₁-C₄-alkyl and heteroaryl-C₂-C₆-alkenyl        groups with 3 to 8 carbon atoms in the heteroaryl group and one        or two hetero-atom(s) selected from N, O and S, which may be        substituted with C₁-C₆ alkyl and/or C₂-C₆ alkoxy groups;        with a free functional group of the underlying transport        mediator T and at least one free functional group of the        underlying colloid P, which are independently selected from    -   amino group (—NH₂); or    -   hydroxy group (—OH)        to form the linker Z, wherein said colloid P and/or transport        mediator T is linked with m units -(L-A).

In addition, in a further preferred embodiment, the compounds accordingto the invention are obtainable by the reaction of a diester of generalformula VI

R⁵(—COOR′)₂  (VI)

wherein R′ is a C₁₋₁₀ alkyl group and R⁵ is selected from

-   -   a single bond;    -   linear or branched, saturated or unsaturated, aliphatic or        alicyclic hydrocarbyl groups with 1 to 22 carbon atoms;    -   aryl, aryl-C₁-C₄-alkyl and aryl-C₂-C₆-alkenyl groups with 5 to        12 carbon atoms in the aryl group, which may optionally be        substituted with C₁-C₆ alkyl and/or C₂-C₆ alkoxy groups; or    -   heteroaryl, heteroaryl-C₁-C₄-alkyl and heteroaryl-C₂-C₆-alkenyl        groups with 3 to 8 carbon atoms in the heteroaryl group and one        or two hetero-atom(s) selected from N, O and S, which may be        substituted with C₁-C₆ alkyl and/or C₂-C₆ alkoxy groups;        with respectively one free functional group of the underlying        transport mediator T and at least one free functional group of        the underlying colloid P, which are independently selected from    -   amino group (—NH₂); or    -   hydroxy group (—OH)        to form the linker Z, wherein said colloid P and/or transport        mediator T is linked with m units -(L-A).

More preferably, the compounds according to the invention are obtainableby the reaction of a diisocyanate of general formula VII

R⁶(—NCO)₂  (VII)

wherein R⁶ is selected from

-   -   linear or branched, saturated or unsaturated, aliphatic or        alicyclic hydrocarbyl groups with 1 to 22 carbon atoms;    -   aryl, aryl-C₁-C₄-alkyl and aryl-C₂-C₆-alkenyl groups with 5 to        12 carbon atoms in the aryl group, which may optionally be        substituted with C₁-C₆ alkyl and/or C₂-C₆ alkoxy groups; or    -   heteroaryl, heteroaryl-C₁-C₄-alkyl and heteroaryl-C₂-C₆-alkenyl        groups with 3 to 8 carbon atoms in the heteroaryl group and one        or two hetero-atom(s) selected from N, O and S, which may be        substituted with C₁-C₆ alkyl and/or C₂-C₆ alkoxy groups;        with respectively one free functional group of the underlying        transport mediator T and at least one free functional group of        the underlying colloid P, which are independently selected from    -   amino group (—NH₂); or    -   hydroxy group (—OH)        to form the linker Z, wherein said colloid P and/or transport        mediator T is linked with m units -(L-A).

Suitable diisocyanates include, for example, toluoylene diisocyanate,bitoluoylene diisocyanate, dianisidine diisocyanate, tetramethylenediisocyanate, hexamethylene diisocyanate, m-phenylene diisocyanate,m-xylylene diisocyanate, C₁-C₆ alkylbenzene diisocyanate,1-chlorobenzene 2,4-diisocyanate, cyclohexylmethane diisocyanate,3,3′-dimethoxydiphenylmethane 4,4′-diisocyanate, 1-nitrobenzene2,4-diisocyanate, 1-alkoxybenzene 2,4-diisocyanate, ethylenediisocyanate, propylene diisocyanate, cyclohexylene 1,2-diisocyanate,3,3′-dichloro-4,4′-biphenylene diisocyanate, diphenylene diisocyanate,2-chlorotrimethylene diisocyanate, butylene 1,2-diisocyanate, ethylidenediisocyanate, diphenylmethane 4,4′-diisocyanate, diphenylethanediisocyanate, 1,5-naphthalene diisocyanate, cyclohexane diisocyanate andisophorone diisocyanate.

More preferably, the compound according to the invention is obtainableby the reaction of a diepoxide with respectively one free functionalgroup of the underlying transport mediator T and at least one freefunctional group of the underlying colloid P, which are independentlyselected from

-   -   amino group (—NH₂); or    -   hydroxy group (—OH)        to form the linker Z, wherein said colloid P and/or transport        mediator T is linked with m units -(L-A).

In particular, 1,2,3,4-diepoxybutane or 1,2,7,8-diepoxyoctane haveproven to be suitable diepoxides.

Compounds in which the linking of the transport mediator T and colloid Pis effected by reductive amination have proven particularlyadvantageous. Thus, more preferably, the compounds according to theinvention are obtainable by reductive amination of a colloid P havingfree amino groups (—NH₂) with a transport mediator T having at least onealdehyde or keto group, and wherein the colloid P and/or transportmediator T is linked with m units -(L-A).

Herein, the colloid P having amino groups is preferably selected fromthe group consisting of aminated starch, aminated hydroxyalkyl starch,aminated hydroxyalkyl carboxyalkyl starch, and aminated carboxyalkylstarch. Particularly preferred is aminated hydroxyalkyl starch, whichmay itself be obtained, for example, by reductive amination.

More preferably, the colloids P linked by reductive amination withtransport mediators T include transport mediators selected from thegroup of heparin or heparin derivatives.

In a particularly preferred embodiment of the present invention, thetransport mediator T is heparin, and the colloid P is a hydroxyethylstarch, and the first linker Z is an —NH group.

As already set forth above, the second linker L is preferably afunctional group selected from carboxylic acid ester, carboxylic acidamide, urethane, ether and amine groups or comprises at least one suchfunctional group.

Further, several colloids P may also be linked through the linkermolecules L and/or Z to form larger clusters. This reaction may competewith the binding of A and/or T to the colloid P. According to theinvention, the ratio of these competing reactions can be influenced bysuitably modifying the process employed. This can be done most simply bychanging the ratio of reagents and substrates employed and by modifyingthe molecular weight of the colloid. Further, reaction conditions suchas the temperature, pressure and catalysts also influence the ratio ofthe two reactions.

The colloid P may have one or more transport mediators T linked throughthe first linker Z. The number of transport mediators T linked with thecolloid P is defined by the parameter n. In a preferred embodiment ofthe present invention, n is an integer of from 1 to 10,000, preferablyfrom 2 to 1000, more preferably from 5 to 500, especially from 10 to100.

In a further preferred embodiment of the present invention, thetransport mediator T and/or the colloid P is covalently linked throughthe second linker L with A, i.e., the medicinally active substance orfluorescence marker. Therefore, in a preferred embodiment of the presentinvention, the parameter m is an integer of at least 1, preferably m isan integer of from 1 to 10,000, more preferably from 2 to 1000, evenmore preferably from 5 to 500, especially from 10 to 100.

The present invention further relates to a pharmaceutical formulationcomprising the compound according to the invention.

The pharmaceutical formulation according to the invention is preferablyan aqueous formulation and more preferably an injectable one.Preferably, the compound according to the invention is in aconcentration of from 0.0001 to 50% by weight, especially from 0.01 to10% by weight, for example, from 0.1 to 5.5% by weight, respectivelybased on the total composition.

The present invention further relates to a process for preparing thecompound according to the invention by linking a transport mediator Twith a colloid-active compound P to form a linker Z through which T andP are covalently linked with one another, and wherein the colloid Pand/or the transport mediator T is linked with m units -(L-A). Themeanings of T, P, Z, L and A are the same as defined above.

In a preferred embodiment of the process according to the invention, thelinking of the transport mediator T and colloid P is effected byreductive amination.

Herein, it is preferred that, in a first step, the colloid P, which isselected from the group consisting of aminated starch, aminatedhydroxyalkyl starch, aminated hydroxycarboxyalkyl starch, and aminatedcarboxyalkyl starch, is reacted with a transport mediator T selectedfrom the group of heparins or heparin derivatives in the presence of areducing agent.

Preferably, the reducing agent is selected from the group consisting ofLiAlH₄, LiBH₄, NaBH₄ and NaBCNH₃.

In another preferred embodiment of the preparation process according tothe invention, a reductive amination of a modified starch, preferably ahydroxyalkyl starch or a carboxymethyl starch, is first effected in apreliminary step. The reductive amination is advantageously effectedwith ammonia or ammonium hydroxide in the presence of a catalyst. Thisreaction is preferably effected in a hydrogen atmosphere under elevatedpressure, for example, from 10 to 300 bar, preferably from 20 to 100bar, and at temperatures within a range of from 50 to 300° C.,preferably from 80 to 200° C. Raney nickel or cobalt/nickel catalystsare used as catalysts.

The thus obtainable aminated modified starch may subsequently be linkedwith a transport mediator, for example, a glucosaminoglucan, in anotherreductive amination reaction.

In another embodiment of the process of the present invention, thetransport mediator T, preferably the glucosaminoglucan, especially theheparin or the heparin derivative, is applied to an electrically chargedsupport in a first step. Subsequently, the further coupling reactionwith the colloid P is performed, wherein the transport mediator T isleft on the charged support. Applying the transport mediator to thecharged support has proven advantageous, since the ionically chargedregions of the transport mediator T, especially of the heparin or aheparin derivative, will orient themselves towards the charge carrier,and therefore such regions are hardly accessible for the couplingreaction with the colloid P. Thus, specific binding domains of thetransport mediator can be selectively obtained with this process.

In a particular embodiment of the present invention, heparin or aheparin derivative is employed as the transport mediator, andhydroxyethyl starch or carboxymethyl starch is employed as the colloid.

Preferably, the part of the heparin molecule intended for association tothe heparin binding domain is associated with a body, preferably ananostructured body, more preferably a positively charged one, and keptfree from the covalent bonding into the hydroxyethyl starch and/orcarboxymethyl starch molecule. For example, for this purpose, a copperplate may be coated with an insulator and freed from the insulator layerat selected sites by using a laser. It is particularly advantageous ifthe charge structures of the heparin binding domains are detected byscanning electron microscopy, and corresponding charge patterns for theimmobilization of the heparin molecules are fired into the insulatorlayer. Also, the scanning electron microscopy can be used forintroducing suitable charge patterns into the insulator layer of theassociation body. Corresponding applications of laser technology areknown to the skilled person. The application of the transport mediatorto a charged surface may also be effected directly by application toother positively charged molecules. Particularly suitable for thispurpose are strongly positively charged polymers that are present in theform of a film as a polycation under the reaction conditions of thesyntheses according to the invention. In linkers reacting under alkalinereaction conditions, chitosan, for example, is present as a polycation,to which heparin is readily associated. It is to be noted that when theheparin is covalently bound to the colloid P, especially hydroxyethylstarch, a linkage is not to be formed between a functional group of thechitosan and the polysaccharide, but only between the hydroxyethylstarch and the heparin.

The invention will be further illustrated by the following Examples, butwithout being limited thereto.

EXAMPLES Example 1a Linking of Fluorescein Isothiocyanate CoupledHydroxyethyl Starch (FITC-HES) with Heparin

200 mg of heparin¹⁾ is dissolved in 10 ml of PBS (phosphate-bufferedsaline), pH 7.5, and the solution is pipetted onto a plate positivelycharged with a Van de Graaff generator. The plate is kept charged for 1week until the solution has dried as a film. Onto the film, 0.2 ml of1,2,7,8-diepoxyoctane (from ALFA-AESAR GmbH & Co. KG, Germany) ispipetted and distributed by rotation. According to the method describedby DeBelder and Granath²⁾, 80 mg of hydroxyethyl starch (HES) [averagemolecular weight: 50 kDa; DS=0.4] is covalently bound to fluoresceinisothiocyanate units (FITC-HES). The FITC-HES is dissolved in 10 ml of amixture of 3 ml of 1 N NaOH and 7 ml of acetone, and dropped onto thecharged plate with shaking. The mixture is adjusted to a pH of 10 andshaken every 30 minutes in a darkened room. After 12 hours, the solutionis withdrawn, dialyzed against distilled water and subsequentlyfreeze-dried. The reagent is taken up in 10 ml of PBS, pH=7.5. In anelectrophoresis, the linking product migrates significantly faster thana FITC-HES not linked to heparin. ¹⁾ Heparin (also abbreviated as HEP):the sodium salt was employed (of porcine origin), pH=7, averageM_(w)=12-15 kDa, manufacturer: Changzhou Qianhong Bio-Pharma Co., Ltd.,Jiangsu, China.²⁾ A. N. De Belder and Kirsti Granath; CarbohydrateResearch, 30 (1973), 375-378.

Example 1 b) Effectiveness of the FITC-HES-Heparin Compound According toExample 1a)

100 mg of dry substance of the FITC-HES-heparin compound synthesized inExample 1a) is dissolved in 5 ml of an aqueous 0.9% NaCl solution, andthe solution is injected i.p. into a Wistar rat. After 6 hours, theanimal was sacrificed under anesthesia, and the organs were removed.

From the spleen, a piece of tissue sized 0.6×0.8 cm was taken and placedin formalin over night. After an ascending ethanol series and methylbenzoate series, sections having a thickness of 6-8 μm were prepared.The preparations were observed with a fluorescence microscope at awavelength of 450-490 nm.

FIGS. 1 and 2 show a strong fluorescence of the cells at 20 timesmagnification. The bright areas in the photographs demonstrate theuptake of the fluorescence-marked HES-heparin complex into the tissuecells of the spleen.

Example 2 Linking of Carboxymethyl Hydroxyethyl Starch with Heparin

200 mg of heparin¹⁾ is dissolved in distilled water and treated like inExample 1a). 10 ml of a 6% carboxymethyl hydroxyethyl starch [DS forcarboxymethyl groups=0.06, and DS for the hydroxyethyl groups=0.34] isdissolved in 10 ml of a 0.1 N HCl acetone solution (3 ml of 0.1 N HCland 7 ml of acetone) and added together with 0.2 ml1,2,7,8-diepoxyoctane, followed by shaking.

The mixture is adjusted to a pH of 3 to 4 by adding the HCl/acetonesolution and shaken every 30 minutes. After 12 hours, the solution iswithdrawn, dialyzed against distilled water and subsequentlyfreeze-dried.

Example 3 Linking of an Aminated HES with Heparin

200 g of a hydroxyethyl starch (HES) [average molecular weightMw=50,000; DS=0.3] is placed into an autoclave together with a 27%ammonium hydroxide solution and 300 g of a nickel/copper/chromiumcatalyst with a nickel proportion of 75%, a copper proportion of 23% anda chromium proportion of 2%. Under addition of hydrogen, the autoclaveis pressurized over a period of 12 hours. The temperature is adjusted to220° C. Subsequently, the mixture is withdrawn, dialyzed andfreeze-dried. 200 mg of heparin¹⁾ is dissolved in 5 ml of PBS, pH=−7.5,and pipetted onto a Petri dish positively charged with a Van de Graaffgenerator. 200 mg of the reductively aminated hydroxyethyl starch isdissolved in 10 ml of distilled water, and the solution is carefullyadded. Thereafter, 0.025 mg of sodium cyanoborohydride NaBH₃CN isadmixed. The Petri dish is carefully shaken. After 2 hours, again 0.025mg of the sodium cyanoborohydride is added, and the mixture is carefullyshaken until bubbles cease to rise. The addition of sodiumcyanoborohydride is repeated four times in the same way. Thereafter, thereagent is allowed to stand for 72 hours; finally, it is taken up in anexcess of PBS, pH=7.5, dialyzed and freeze-dried.

Example 4 Linking of an Aminated HES with Heparin, Followed by Reactionwith Human Albumin

200 g of a hydroxyethyl starch (HES) [average molecular weightMw=50,000; DS=0.3] is placed into an autoclave together with a 27%ammonium hydroxide solution and 300 g of a nickel/copper/chromiumcatalyst with a nickel proportion of 75%, a copper proportion of 23% anda chromium proportion of 2%. Under addition of hydrogen, the autoclaveis pressurized over a period of 12 hours. The temperature is adjusted to270° C. Subsequently, the mixture is withdrawn, dialyzed andfreeze-dried. 200 mg of heparin¹⁾ is dissolved in 5 ml of PBS, pH=7.5,and pipetted onto a Petri dish positively charged with a Van de Graaffgenerator. 200 mg of the reductively aminated hydroxyethyl starch isdissolved in 10 ml of distilled water, and the solution is carefullyadded. Thereafter, 0.025 mg of sodium cyanoborohydride NaBH₃CN isadmixed. The Petri dish is carefully shaken. After 2 hours, again 0.025mg of the sodium cyanoborohydride is added, and the mixture is carefullyshaken until bubbles cease to rise. The addition of sodiumcyanoborohydride is repeated four times in the same way.

Thereafter, the reagent is allowed to stand for 24 hours. After renewedcharging by the Van de Graaff generator, 10 mg of human albumin in 10 mlof PBS (pH=7.5) is added. Subsequently, 0.025 mg of sodiumcyanoborohydride NaBH₃CN is added. The Petri dish is carefully shaken.The addition of 0.025 mg of sodium cyanoborohydride NaBH₃CN followed byshaking is repeated four times with and four times without an operatingVan de Graaff generator. The reagent is finally taken up in an excess ofPBS (pH 7,5), dialyzed and freeze-dried.

Example 5 Linking of Hydroxyethyl Starch with Heparin Throughhexane-1,6-diamine a) Tosylation of the Hydroxyethyl Starch

HES₄₀ ³⁾ (20 g) is suspended in pyridine (200 ml) and heated underreflux until a clear solution has formed. Thereafter, the solution iscooled down to 0° C., and tosyl chloride (19.4 g, 200 equ.) is added inportions with stirring, and the reaction solution is allowed to slowlywarm up to room temperature. With stirring, the reaction solution isadded to acetonitrile (500 ml). Immediately, a white precipitate forms,which is filtered off and dried under vacuum. The white foam obtained isco-evaporated with acetonitrile three times, the residue is taken up isdistilled water, and dialyzed for 24 hours. After removing the water byevaporation, the title compound is obtained as a colorless solid (2.6g). As compared to the pure HES₄₀, the ¹H-NMR spectrum (400 MHz, D₂O)additionally shows typical symmetrical aromatic CH peaks with a chemicalshift of 7-8 ppm, which indicate tosyl groups. ^(3) HES) ₄₀:Hydroxyethyl starch having an average molecular weight M_(w)=40 kDa,degree of substitution DS=0.3; manufacturer: BBraun, Crissier,Switzerland.

b) Substitution of the Tosylated HES with Amino Linker

A solution of 2305-BA-100 (3.3 g) and hexanediamine (10.0 g, 1000 equ.)in DMF (5 ml) is stirred at 50° C. over night and then poured ontoacetone (300 ml). The precipitated solid is filtered off and dried. Forfurther purification, the raw product is dissolved in distilled waterand dialyzed for 24 hours. After removing the water by evaporation, theabove mentioned reaction product is obtained as a colorless solid (1.5g). As compared to heparin, the ¹H-NMR spectrum (400 MHz, D₂O)additionally shows typical CH₂ peaks with a chemical shift of 1-2 ppm,which indicate the amino linker.

c) EDC⁴⁾ Coupling of the Reaction Product Obtained in Step b) with HEP¹⁾⁴⁾ EDC: N-dimethylaminopropyl-N-ethylcarbodiimide hydrochloride

To a solution of HEP (60 mg) and the product obtained in step b) (200mg) in distilled water (4 ml), EDC⁴⁾ hydrochloride (80 mg, 100 equ.) isadded. The reaction solution is stirred at room temperature over nightand thereafter poured onto acetone (5 ml). The precipitated solid isfiltered off and dried. For further purification, the raw product isdissolved in distilled water and dialyzed for 24 hours. After removingthe water by evaporation, the linking product of HES and heparin asshown in the reaction scheme is obtained as a colorless solid (0.11 g).

Example 6 Linking of Hydroxyethyl Starch with Heparin Throughhexane-1,6-diamine

a) EDC⁴⁾ Coupling of Heparin (HEP) with Amino Linker

To a solution of HEP (1.0 g) and hexane-1,6-diamine (0.8 g, 100 equ.) indistilled water (10 ml), EDC⁴⁾ hydrochloride (14 g, 100 equ.) is added.The reaction solution is stirred at 20° C. over night and then pouredonto acetone (20 ml). The precipitated solid is filtered off and dried.By means of LC-MS, it is determined that unreacted hexanediamine iscontained in the reaction product. For further purification, the rawproduct is dissolved in distilled water and dialyzed for 24 hours. Afterremoving the water by evaporation, the coupling product shown in thereaction scheme is obtained as a colorless solid (0.8 g). As compared toheparin, the ¹H-NMR spectrum (400 MHz, D₂O) additionally shows typicalCH₂ peaks with a chemical shift of 1-2 ppm.

b) Nucleophilic Substitution of the Coupling Product Obtained in Step a)with the Tosylated HES from Example 5, Step a)

To a suspension of 2305-AA-1 (30 mg) and 2305-BA100 (100 mg, MW: about50 kDa) in DMSO (4 ml), Et₃N (0.003 ml, 100 equ.) is injected, followedby heating at 80° C. with stirring. The reaction mixture is stirred for6 hours and thereafter poured onto acetone (6 ml). The precipitatedsolid is filtered off and dried. The title compound is obtained as aslightly beige solid (0.1 g). As compared to heparin, the ¹H-NMRspectrum (400 MHz, D₂O) additionally shows typical CH₂ peaks with achemical shift of 1-2 ppm.

Example 7 Linking of an Aminated Hydroxyethyl Starch with Heparin byReductive Amination) a) Amination of the Hydroxyethyl Starch (HES)

HES₄₀ (5.1 g, MW: 40 kDa) is dissolved in an aqueous ammonium hydroxidesolution (100 ml, 22%). The catalyst consisting of nickel (5.6 g, 325mesh), chromium (0.15 g, 100 mesh) and copper (1.8 g, 1 μm) is added tothe solution. The mixture is stirred under a hydrogen atmosphere at 120°C. in an autoclave for 48 hours. After cooling to 20° C., the catalystis filtered off, and the filtrate is poured onto ethanol (20 ml). Theprecipitated solid is filtered off, washed with little ethanol/water,and dried. The aminated HES is obtained as a slightly bluish solid (1.2g).

b) Reductive Amination of the Aminated HES Obtained in Step a) withHeparin

HEP (200 mg) is dissolved in an aqueous phosphate buffer solution (5 ml,pH=7.5), and a solution of the aminated hydroxyethyl starch from step a)(200 mg) in distilled water (10 ml) is added dropwise. At intervals of 2hours, NaCNBH₃ is added six times (0.025 mg each, from an aqueous stocksolution) to the reaction solution. The reaction mixture is againstirred at 20° C. for 2 hours. For further purification, the raw productis dialyzed for 24 hours. After removing the water by evaporation, thelinking product of heparin and aminated hydroxyethyl starch as shown inthe reaction scheme is obtained as a colorless solid (250 mg).

Example 8 Linking of a Fluorescence-Marked Heparin with an AminatedHydroxyethyl Starch (HES) by Reductive Amination

a) Coupling of Heparin (HEP) with the Fluorescence Marker2-aminopyridine

To a solution of 2-aminopyridine (31.7 g, 0.33 mol, 1000 equ.) andNaCNBH₃ (2.1 g, 0.033 mol, 100 equ.) in formamide (50 ml), heparin (5.0g) is added. The suspension obtained is stirred at 37° C. over night,and a clear solution is slowly formed. The reaction solution is pouredonto EtOH (50 ml). The precipitated solid is filtered off and dried. Thecoupling product (HEP*) shown in the reaction scheme is obtained as aslightly beige solid (1.3 g). Both in aqueous solution and as a solid,the coupling product shows an intensive blue-purple fluorescence whenirradiated with UV light at 366 nm. As compared to heparin, the ¹H-NMRspectrum (400 MHz, D₂O) additionally shows typical aromatic CH peakswith a chemical shift of 6.6-7.8 ppm, which indicate typical pyridinesubstituents.

b) Linking of the fluorescence-marked heparin (HEP*) prepared in step a)with the aminated hydroxyethyl starch prepared in Example 7, step a), byreductive amination

HEP* from step a) (200 mg, average molecular weight: 15 kDa) isdissolved in an aqueous phosphate buffer solution (5 ml, pH=7.5), and asolution of the aminated HES from Example 7, step a) (200 mg), indistilled water (10 ml) is added dropwise. At intervals of 2 hours,NaCNBH₃ is added three times (0.025 mg each, from an aqueous stocksolution) to the reaction solution. The reaction mixture is againstirred at 20° C. for 2 hours. For further purification, the raw productis dialyzed for 24 hours. After removing the water by evaporation, thelinking product of aminated HES and fluorescence-marked heparin isobtained as a colorless solid (200 mg).

Both in aqueous solution and as a solid, the compound shows an intensivegreen-yellow fluorescence when irradiated with UV light at 366 nm. Ascompared to heparin, the ¹H-NMR spectrum (400 MHz, D₂O) additionallyshows typical aromatic CH peaks with a chemical shift of 7.0-7.8 ppm,which indicate typical pyridine substituents.

Example 9 Linking of the Product Obtained in Example 5, Step b), withthe Fluorescence-Marked Heparin from Example 8, Step a) by Couplingusing EDC⁴⁾

To a solution of HEP* (Example 8, step a)) (60 mg) and the reactionproduct from Example 5, step b) (160 mg), in distilled water (4 ml), EDChydrochloride (80 mg, 100 equ.) is added. The reaction solution isstirred at 20° C. over night and thereafter poured onto acetone (5 ml).The precipitated solid is filtered off and dried. For furtherpurification, the raw product is dissolved in distilled water anddialyzed for 24 hours. After removing the water by evaporation, thedesired linking product according to the formula scheme shown above isobtained as a colorless solid (0.1 g).

Both in aqueous solution and as a solid, the compound shows an intensivegreen-yellow fluorescence when irradiated with UV light at 366 nm. Ascompared to heparin, the ¹H-NMR spectrum (400 MHz, D₂O) additionallyshows typical aromatic CH peaks with a chemical shift of 7.0-7.8 ppm,which indicate typical pyridine substituents.

1. A compound of general formula (I)(T-Z)_(n)—P  (I), wherein T is a transport mediator; P is acolloid-active compound; Z is a first linker by means of which T and Pare covalently linked together; and n is an integer of at least 1; andwherein the transport mediator T and/or the colloid P bears m groups-(L-A), wherein A is a medicinally active substance or a fluorescencemarker; L is a second linker through which P is covalently linked withA, or through which T is covalently linked with A; and m is an integerthat is 0 or at least
 1. 2. The compound according to claim 1,characterized in that said transport mediator has at least one bindingsite for association to cellular binding domains.
 3. The compoundaccording to claim 1, characterized in that said transport mediator T isselected from the group consisting of sialic acid, polysialic acid,neuraminic acid, N-acetylneuraminic acid, mannose, N-acetylmannose,N-propanolmannosamine, fucose, N-acetylfucose, galactose,N-acetylgalactose, glucose, N-acetylglucose, hexoses, N-acetylhexoses,ceramides, glucose-6-phosphate, mannose-6-phosphate,glucosylphosphatidylinositol, retinic acid, immunoglobulins,monoglycerates, diacylglycerates, sphingomyelin, bisphosphonates,glycoproteins, and glycosaminoglycans.
 4. The compound according toclaim 1, characterized in that said transport mediator T is aglycosaminoglycan or a glycosaminoglycan derivative, which arepreferably selected from the group consisting of heparin and heparinsulfate, especially heparin or heparin sulfate having less than 6saccharide units.
 5. The compound according to claim 1, characterized inthat said colloid-active compound P is selected from the groupconsisting of amyloses, amylopectins, acemannans, arabinogalactans,galactomannans, galactoglucomannans, xanthans, carrageenan, starch andmodified starch.
 6. The compound according to claim 5, characterized inthat said modified starch is selected from the group consisting ofhydroxyalkyl starches, esterified starches, carboxyalkyl starches,hydroxyalkyl carboxyalkyl starch, aminated hydroxyalkyl starch, aminatedhydroxyalkyl carboxyalkyl starch and aminated carboxyalkyl starch. 7.The compound according to claim 6, characterized in that said modifiedstarch is selected from hydroxyethyl starch or aminated hydroxyethylstarch.
 8. The compound according to claim 5, characterized in that saidcolloid-active compound has an average molecular weight of from 20,000to 800,000 daltons, preferably from 25,000 to 500,000 daltons,especially from 30,000 to 200,000 daltons.
 9. The compound according toclaim 5, characterized in that the degree of substitution, DS, of themodified starch, especially hydroxyethyl starch, is from 0.2 to 0.8,preferably from 0.3 to 0.6.
 10. The compound according to claim 1,characterized in that said medicinally active compound A is selectedfrom the group consisting of antibiotics, chemotherapeutics, cytostaticagents, antigens, oligonucleotides, mediators, false metabolicsubstrates, analgetics and cytotoxic substances.
 11. The compoundaccording to claim 1, characterized in that said fluorescence marker isselected from the group consisting of fluorescein isothiocyanate (FITC),phycoerythrin, rhodamide and 2-aminopyridine.
 12. The compound accordingto claim 1, characterized in that said linker Z is a functional groupselected from carboxylic acid ester, carboxylic acid amides, urethane,ether and amine groups, or comprises such a group.
 13. The compoundaccording to claim 1, obtainable by a reaction of at least one freeisocyanate group (—NCO); carboxy group (—COOH); carboxylic acid halidegroup (—CO-A, with A=Cl, Br or I); alkylenecarboxy group(—(CH₂)_(q)—COOH, with q=1-10); ester group (—COOR with R=organicradical); epoxy group; or nucleophilic leaving group; of the underlyingcolloid P with a free hydroxy group (—OH) of the underlying transportmediator T to form the linker Z, wherein said colloid P and/or transportmediator T is linked with m units -(L-A).
 14. The compound according toclaim 1, obtainable by a reaction of at least one free hydroxy group(—OH) of the underlying colloid P with a free isocyanate group (—NCO);carboxy group (—COON); carboxylic acid halide group (—CO-A, with A=Cl,Br or I); alkylenecarboxy group (—(CH₂)_(q)—COON, with q=1-10); estergroup (—COOR with R=organic radical); epoxy group; or nucleophilicleaving group; of the underlying transport mediator T to form the linkerZ, wherein said colloid P and/or transport mediator T is linked with munits -(L-A).
 15. The compound according to claim 1, obtainable by areaction of at least one free amino group (—NH₂) of the underlyingcolloid P with a free isocyanate group (—NCO); carboxy group (—COOH);carboxylic acid halide group (—CO-A, with A=CI, Br or I);alkylenecarboxy group (—(CH₂)_(q)—COOH, with q=1-10); ester group (—COORwith R=organic radical); epoxy group; or nucleophilic leaving group; ofthe underlying transport mediator T to form the linker Z, wherein saidcolloid P and/or transport mediator T is linked with m units -(L-A). 16.The compound according to claim 1, obtainable by a reaction of at leastone free isocyanate group (—NCO); carboxy group (—COOK); carboxylic acidhalide group (—CO-A, with A=Cl, Br or I); alkylenecarboxy group(—(CH₂)_(q)—COOH, with q=1-10); ester group (—COOR with R=organicradical); epoxy group; or nucleophilic leaving group; of the underlyingcolloid P with a free amino group (—NH₂) of the underlying transportmediator T to form the linker Z, wherein said colloid P and/or transportmediator T is linked with m units -(L-A).
 17. The compound according toclaim 1, obtainable by a reaction of at least one free hydroxy group(—OH); or amino group (—NH₂) of the underlying colloid P with a freeisocyanate group (—NCO); carboxy group (—COON); carboxylic acid halidegroup (—CO-A, with A=Cl, Br or I); alkylenecarboxy group(—(CH₂)_(q)—COOH, with q=1-10); ester group (—COOR with R=organicradical); epoxy group; or nucleophilic leaving group; of the underlyingtransport mediator T to form the linker Z, wherein said colloid P and/ortransport mediator T is linked with m units -(L-A).
 18. The compoundaccording to claim 1, obtainable by a reaction of a diamine of generalformula IIR¹(—NH₂)₂  (II) wherein R¹ is selected from a single bond; linear orbranched, saturated or unsaturated, aliphatic or alicyclic hydrocarbylgroups with 1 to 22 carbon atoms; aryl, aryl-C₁-C₄-alkyl andaryl-C₂-C₆-alkenyl groups with 5 to 12 carbon atoms in the aryl group,which may optionally be substituted with C₁-C₆ alkyl and/or C₂-C₆ alkoxygroups; or heteroaryl, heteroaryl-C₁-C₄-alkyl andheteroaryl-C₂-C₆-alkenyl groups with 3 to 8 carbon atoms in theheteroaryl group and one or two hetero-atom(s) selected from N, O and S,which may be substituted with C₁-C₆ alkyl and/or C₂-C₆ alkoxy groups;with a free functional group of the underlying transport mediator T andat least one free functional group of the underlying colloid P, whichare independently selected from isocyanate group (—NCO); carboxy group(—COOH); carboxylic acid halide group (—CO-A, with A=Cl, Br or I);alkylenecarboxy group (—(CH₂)_(q)—COOH, with q=1-10); ester group (—COORwith R=organic radical); epoxy group; or nucleophilic leaving group; toform the linker Z, wherein said colloid P and/or transport mediator T islinked with m units -(L-A).
 19. The compound according to claim 1,obtainable by a reaction of a diol of general formula IIIR²(—OH)₂  (III), wherein R² is selected from linear or branched,saturated or unsaturated, aliphatic or alicyclic hydrocarbyl groups with2 to 22 carbon atoms; aryl, aryl-C₁-C₄-alkyl and aryl-C₂-C₆-alkenylgroups with 5 to 12 carbon atoms in the aryl group, which may optionallybe substituted with C₁-C₆ alkyl and/or C₂-C₆ alkoxy groups; orheteroaryl, heteroaryl-C₁-C₄-alkyl and heteroaryl-C₂-C₆-alkenyl groupswith 3 to 8 carbon atoms in the heteroaryl group and one or twohetero-atom(s) selected from N, O and S, which may be substituted withC₁-C₆ alkyl and/or C₂-C₆ alkoxy groups; with a free functional group ofthe underlying transport mediator T and at least one free functionalgroup of the underlying colloid P, which are independently selected fromisocyanate group (—NCO); carboxy group (—COOH); carboxylic acid halidegroup (—CO-A, with A=Cl, Br or I); alkylenecarboxy group(—(CH₂)_(q)—COOH, with q=1-10); ester group (—COOR with R=organicradical); epoxy group; or nucleophilic leaving group; to form the linkerZ, wherein said colloid P and/or transport mediator T is linked with munits -(L-A).
 20. The compound according to claim 1, obtainable by areaction of a dicarboxylic acid of general formula IVR³(—COOH)₂  (IV) wherein R³ is selected from a single bond; linear orbranched, saturated or unsaturated, aliphatic or alicyclic hydrocarbylgroups with 1 to 22 carbon atoms; aryl, aryl-C₁-C₄-alkyl andaryl-C₂-C₆-alkenyl groups with 5 to 12 carbon atoms in the aryl group,which may optionally be substituted with C₁-C₆ alkyl and/or C₂-C₆ alkoxygroups; or heteroaryl, heteroaryl-C₁-C₄-alkyl andheteroaryl-C₂-C₆-alkenyl groups with 3 to 8 carbon atoms in theheteroaryl group and one or two hetero-atom(s) selected from N, O and S,which may be substituted with C₁-C₆ alkyl and/or C₂-C₆ alkoxy groups;with a free functional group of the underlying transport mediator T andat least one free functional group of the underlying colloid P, whichare independently selected from amino group (—NH₂); or hydroxy group(—OH) to form the linker Z, wherein said colloid P and/or transportmediator T is linked with m units -(L-A).
 21. The compound according toclaim 1, obtainable by a reaction of a dicarboxylic acid halide ofgeneral formula VR⁴(—CO-A)₂  (V) wherein A=Cl, Br or I, and R⁴ is selected from a singlebond; linear or branched, saturated or unsaturated, aliphatic oralicyclic hydrocarbyl groups with 1 to 22 carbon atoms; aryl,aryl-C₁-C₄-alkyl and aryl-C₂-C₆-alkenyl groups with 5 to 12 carbon atomsin the aryl group, which may optionally be substituted with C₁-C₆ alkyland/or C₂-C₆ alkoxy groups; or heteroaryl, heteroaryl-C₁-C₄-alkyl andheteroaryl-C₂-C₆-alkenyl groups with 3 to 8 carbon atoms in theheteroaryl group and one or two hetero-atom(s) selected from N, O and S,which may be substituted with C₁-C₆ alkyl and/or C₂-C₆ alkoxy groups;with a free functional group of the underlying transport mediator T andat least one free functional group of the underlying colloid P, whichare independently selected from amino group (—NH₂); or hydroxy group(—OH) to form the linker Z, wherein said colloid P and/or transportmediator T is linked with m units -(L-A).
 22. The compound according toclaim 1, obtainable by a reaction of a diester of general formula VIR⁵(—COOR′)₂  (VI) wherein R′ is a C₁₋₁₀ alkyl group and R⁵ is selectedfrom a single bond; linear or branched, saturated or unsaturated,aliphatic or alicyclic hydrocarbyl groups with 1 to 22 carbon atoms;aryl, aryl-C₁-C₄-alkyl and aryl-C₂-C₆-alkenyl groups with 5 to 12 carbonatoms in the aryl group, which may optionally be substituted with C₁-C₆alkyl and/or C₂-C₆ alkoxy groups; or heteroaryl, heteroaryl-C₁-C₄-alkyland heteroaryl-C₂-C₆-alkenyl groups with 3 to 8 carbon atoms in theheteroaryl group and one or two hetero-atom(s) selected from N, O and S,which may be substituted with C₁-C₆ alkyl and/or C₂-C₆ alkoxy groups;with respectively one free functional group of the underlying transportmediator T and at least one free functional group of the underlyingcolloid P, which are independently selected from amino group (—NH₂); orhydroxy group (—OH) to form the linker Z, wherein said colloid P and/ortransport mediator T is linked with m units -(L-A).
 23. The compoundaccording to claim 1, obtainable by a reaction of a diisocyanate ofgeneral formula VIIR⁶(—NCO)₂  (VII) wherein R⁶ is selected from linear or branched,saturated or unsaturated, aliphatic or alicyclic hydrocarbyl groups with1 to 22 carbon atoms; aryl, aryl-C₁-C₄-alkyl and aryl-C₂-C₆-alkenylgroups with 5 to 12 carbon atoms in the aryl group, which may optionallybe substituted with C₁-C₆ alkyl and/or C₂-C₆ alkoxy groups; orheteroaryl, heteroaryl-C₁-C₄-alkyl and heteroaryl-C₂-C₆-alkenyl groupswith 3 to 8 carbon atoms in the heteroaryl group and one or twohetero-atom(s) selected from N, O and S, which may be substituted withC₁-C₆ alkyl and/or C₂-C₆ alkoxy groups; with respectively one freefunctional group of the underlying transport mediator T and at least onefree functional group of the underlying colloid P, which areindependently selected from amino group (—NH₂); or hydroxy group (—OH)to form the linker Z, wherein said colloid P and/or transport mediator Tis linked with m units -(L-A).
 24. The compound according to claim 1,obtainable by a reaction of a diepoxide with respectively one freefunctional group of the underlying transport mediator T and at least onefree functional group of the underlying colloid P, which areindependently selected from amino group (—NH₂); or hydroxy group (—OH)to form the linker Z, wherein said colloid P and/or transport mediator Tis linked with m units -(L-A).
 25. The compound according to claim 1,obtainable by reductive amination of a colloid P having free aminogroups (—NH₂) with a transport mediator T having at least one aldehydeor keto group, and wherein the colloid P and/or transport mediator T islinked with m units -(L-A).
 26. The compound according to claim 25,characterized in that said colloid P having amino groups is selectedfrom the group consisting of aminated starch, aminated hydroxyalkylstarch, aminated hydroxyalkyl carboxyalkyl starch, and aminatedcarboxyalkyl starch.
 27. The compound according to claim 25,characterized in that said transport mediator is heparin or a heparinderivative.
 28. The compound according to claim 1, characterized in thatsaid transport mediator T is heparin, and said colloid P is ahydroxyethyl starch, and the first linker Z is an —NH group.
 29. Thecompound according to claim 1, characterized in that said second linkerL is a functional group selected from carboxylic acid ester, carboxylicacid amide, urethane, ether and amine groups, or comprises such a group.30. A pharmaceutical formulation comprising the compound according toclaim
 1. 31. The pharmaceutical formulation according to claim 30,characterized in that said formulation is aqueous and injectable.
 32. Aprocess for preparing a compound of general formula (I) according toclaim 1, by linking a transport mediator T with a colloid-activecompound P to form a linker Z through which T and P are covalentlylinked with one another, and wherein the colloid P and/or the transportmediator T is linked with m units -(L-A).
 33. The process according toclaim 32, characterized in that the linking of the transport mediator Tand colloid P is effected by reductive amination.
 34. The processaccording to claim 32 or characterized in that, in a first step, thecolloid P, which is selected from the group consisting of aminatedstarch, aminated hydroxyalkyl starch, aminated hydroxycarboxyalkylstarch, and aminated carboxyalkyl starch, is reacted with a transportmediator T selected from the group of heparins or heparin derivatives inthe presence of a reducing agent.
 35. The process according to claim 34,characterized in that the reducing agent is selected from the groupconsisting of LiAlH₄, LiBH₄, NaBH₄ and NaBH₃CN.